The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbß3 reconstituted into physiologically mimetic GUVs.

Platelet integrin αIIbß3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbß3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIbß3 behaviour away from the complexity of the platelet machinery. Here, a novel method of αIIbß3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. αIIbß3 was inserted into two GUV lipid compositions that seek to better mimic the platelet membrane. First, "nature's own", comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn(2+) or DTT does not influence the diffusion coefficient of αIIbß3. Similarly, exposure to αIIbß3's primary ligand fibrinogen (Fg) alone does not affect αIIbß3's diffusion coefficient. However, addition of Fg with either activator reduces the integrin diffusion coefficient from 2.52 ± 0.29 to µm(2) s(-1) to 1.56 ± 0.26 (Mn(2+)) or 1.49 ± 0.41 µm(2) s(-1) (DTT) which is consistent with aggregation of activated αIIbß3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin-Fg complex diffuses through the confocal volume in clusters. Using the Saffman-Delbrück model as a first approximation, the diffusion coefficient of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIbß3 was also reconstituted into a "raft forming" GUV with well defined liquid disordered (Ld) and liquid ordered (Lo) phases. Using confocal microscopy and lipid partitioning dyes, αIIbß3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIbß3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbß3 into cholesterol rich domains is not responsible for the integrin clustering observed.